The objectives of this proposal are to examine the individual roles and interaction of surface membrane flow, substrate attachment, and cytoplasmic contractility in amoeboid locomotion and to isolate mutants defective in motility as a means of analyzing amoeboid movement genetically. Spermatozoa of the nematode, Caenorhabditis elegans will be used for study because these amoeboid cells exhibit a well-defined directed surface membrane flow on their pseudopods and require a specific, naturally-occurring substrate for efficient locomotion. In addition, the extensive genetic background of C. elegans allows easy isolation of mutant sperm altered in motility. Preliminary studies on membrane flow will be extended to examine the rate and spatial organization of membrane internalization, recycling of membrane components through the cytoplasm, and their reinsertion onto the surface. The dynamics of cell-substrate attachment sites and structural association between these sites and cytoplasmic components will be examined by light, scanning and transmission electron microscopy. Both the membrane and substrate molecules involved in attachment will be characterized. Exploratory experiments on demembranated sperm will seek to demonstrate cytoplasmic contraction and to characterize the proteins involved. Antisera to these proteins will be used to compare their distributions in sessile and motile cells. Two sperm mutants with altered motility have already been isolated. Existing sperm-specific mutants will be screened for motility defects and new mutants will be sought. Information gained from studies of wild-type sperm locomotion will be used to examine the motility defects of existing and new mutants in detail. The long-range objective of this work is to determine, in molecular detail, how an amoeboid cell crawls. This goal can be achieved much more readily using a simple experimental animal that can be manipulated genetically than by studying mammalian cells. Amoeboid locomotion and its regulation are key components to understanding cellular migrations during embryogenesis, surveillance and responsiveness in the cellular immune system, and altered regulatory mechanisms that accompanying malignant transformation.